Estrogen combination for treatment of multiple sclerosis

ABSTRACT

Provided are methods for treating multiple sclerosis using a continuous regimen of estrogen in combination with periodic administration of a progestrogen.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/985,184, filed on Apr. 28, 2014, U.S. Provisional PatentApplication No. 61/985,380, filed on Apr. 28, 2014. U.S. ProvisionalPatent Application No. 62/032,952, filed on Aug. 4, 2014, and U.S.Provisional Patent Application No. 62/050,918, filed on Sep. 16, 2014,each of which is hereby incorporated by reference in its entirety.

BACKGROUND

Multiple sclerosis (MS) is a chronic, often debilitating diseaseaffecting the central nervous system (brain and spinal cord). MS affectsmore than 1 million people worldwide and is the most common neurologicaldisease among young adults, particularly women. The exact cause of MS isstill unknown. MS is an autoimmune disease in which myelin sheathssurrounding neuronal axons are destroyed. This condition can causeweakness, impaired vision, loss of balance, and poor musclecoordination.

MS takes several forms, with new symptoms either occurring in isolatedattacks (relapsing forms) or building up over time (progressive forms).Between attacks, symptoms may disappear completely; however, permanentneurological problems often occur, especially as the disease advances.

In 1996, the United States National Multiple Sclerosis Society describedfour clinical subtypes of MS: (i) relapsing-remitting; (ii)secondary-progressive; (iii) primary-progressive; and (iv)progressive-relapsing.

Relapsing-remitting MS is characterized by unpredictable relapsesfollowed by periods of months to years of relative quiet (remission)with no new signs of disease activity. Deficits that occur duringattacks may either resolve or leave sequelae, the latter in about 40% ofattacks and being more common the longer a person has had the disease.This describes the initial course of 80% of individuals with MS. Whendeficits always resolve between attacks, this is sometimes referred toas benign MS, although people will still build up some degree ofdisability in the long term. On the other hand, the term malignantmultiple sclerosis is used to describe people with MS having reachedsignificant level of disability in a short period of time. Therelapsing-remitting subtype usually begins with a clinically isolatedsyndrome (CIS). In CIS, a person has an attack suggestive ofdemyelination but does not fulfill the criteria for multiple sclerosis;30 to 70% of persons experiencing CIS go on to develop MS.

Secondary-progressive MS occurs in around 65% of those with initialrelapsing-remitting MS, who eventually have progressive neurologicdecline between acute attacks without any definite periods of remission.Occasional relapses and minor remissions may appear. The median lengthof time between disease onset and conversion from relapsing-remitting tosecondary progressive MS is 19 years.

Primary-progressive MS occurs in approximately 10-20% of individuals,with no remission after the initial symptoms. It is characterized byprogression of disability from onset, with no, or only occasional andminor, remissions and improvements. The usual age of onset for theprimary progressive subtype is later than of the relapsing-remittingsubtype, but similar to the age that secondary-progressive MS usuallybegins in relapsing-remitting MS, around 40 years of age.

Progressive-relapsing MS describes those individuals who, from onset,have a steady neurologic decline but also have clear superimposedattacks. This is the least common of all subtypes.

Currently the following agents are approved by the U.S. Food and DrugAdministration (FDA) to reduce disease activity and disease progressionfor many people with relapsing forms of MS, includingrelapsing-remitting MS, as well as secondary-progressive andprogressive-relapsing MS in those people who continue to have relapses:dimethyl fumarate (Teefidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®), interferon beta-1a (Avonex® and Rebif®), interferonbeta-1b (Betaseron® and Extavia®), mitoxantrone (Novantrone®),natalizumab (Tysabri®), and teriflunomide (Aubagio®). However, many ofthese therapies fail to successfully treat all patients or all symptomsin treated patients, and many of these therapies are associated withundesirable side effects. Accordingly, alternative therapies are needed.

SUMMARY

An aspect of the invention is a method of treating multiple sclerosis,comprising

administering to a subject in need thereof, on a continuous basisthroughout one or more (preferably at least two) consecutive treatmentperiods, a therapeutically effective amount of an estrogen; and

administering to the subject, for only a portion of each treatmentperiod, a therapeutically effective amount of a progestrogen.

In certain embodiments, the estrogen is selected from estriol (E3),estradiol (E2), estrone (E1), pharmaceutically acceptable salts of anyof the foregoing, and any combination thereof.

In certain embodiments, the estrogen is estriol.

In certain embodiments, the progestrogen is selected from chlormadinoneacetate, cyproterone acetate, desogestrel, dienogest,5α-dihydroprogesterone, drospirenone (Yasmin®), ethinodiol acetate,ethynodiol diacetate, etonogestrel (Nexplanon®), gestodene,17-hydroxyprogesterone, levonorgestrel (Alesse®), medroxyprogesteroneacetate (17α-hydroxy-6α-methylprogesterone acetate; Provera®),megestrol, megestrol acetate(17α-acetoxy-6-dehydro-6-methylprogesterone), nestorone, nomegestrolacetate, norethindrone, norethindrone acetate (also known asnorethisterone acetate), norethynodrel (Enovid®), norgestimate,norgestrel, progesterone, tanaproget, trimegestone, pharmaceuticallyacceptable salts of any of the foregoing, and any combination thereof.

In certain embodiments, the progestrogen is progesterone.

In certain embodiments, the progestrogen is norethindrone.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 8 mg of estriol daily.

In certain embodiments, the progestrogen is administered orally in adose equal or equivalent to about 700 μg of norethindrone daily.

An aspect of the invention is a method of treating multiple sclerosis,comprising administering orally to a subject in need thereof, on acontinuous basis for 84 consecutive days (12 weeks), 8 mg of estrioldaily; and administering orally to the subject, for 14 consecutive days(2 weeks) of the 84 consecutive days (12 weeks), 0.7 mg of norethindronedaily.

In certain embodiments, the method further comprises administering tothe subject a placebo in place of the norethindrone on each of the daysthe norethindrone is not administered to the subject.

In certain embodiments, the multiple sclerosis is relapsing-remittingmultiple sclerosis.

In certain embodiments, the multiple sclerosis is secondary-progressivemultiple sclerosis.

In certain embodiments, the multiple sclerosis is primary-progressivemultiple sclerosis.

In certain embodiments, the multiple sclerosis is progressive-relapsingmultiple sclerosis.

In certain embodiments, the multiple sclerosis is clinically isolatedsyndrome (CIS).

In certain embodiments, the method further comprises administering tothe subject an immunotherapeutic agent, wherein the immunotherapeuticagent is neither an estrogen nor a progestrogen, e.g., animmunotherapeutic agent selected from interferon-beta 1a,interferon-beta 1b, glatiramer acetate, natalizumab, mitoxantrone,fingolimod, teriflunomide, and dimethyl fumarate.

In certain embodiments, the subject is a subject being treated with animmunotherapeutic agent yet experiencing a relapse and/or progression ofthe multiple sclerosis.

Although the methods disclosed throughout the specification and claimsare useful for treating multiple sclerosis in its various forms andstages, these methods can also be applied the treatment of otherneurodegenerative diseases, such as, by way of illustration, Alzheimer'sdisease, Parkinson's disease, stroke, amyotrophic lateral sclerosis,cerebellar ataxia, frontotemporal dementia, prion disease, Huntington'sDisease, cerebral ischemia, idiopathic Morbus Parkinson. Parkinsonsyndrome, Morbus Alzheimers, cerebral dementia syndrome,infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglyemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, degenerative retina disorders,trauma-induced brain damage, trauma-induced bone marrow damage, cerebralhyperexcitability symptoms, cerebral hyperexcitability states (e.g., ofvarying origin, such as after the addition of and/or withdrawal ofmedicaments, toxins, noxae and drugs), neurodegenerative syndromes ofthe peripheral nervous system, peripheral nerve injury, and spinal cordinjury. In certain preferred embodiments, the neurodegenerative diseaseis multiple sclerosis. In preferred embodiments, the patient is a woman.In some embodiments, the patient is a premenopausal or perimenopausalwoman. In other embodiments, the patient is a postmenopausal woman.

In certain embodiments, the method is a method for slowing, halting, orreversing progression of a cognitive or physical disability in a subjectwith a neurodegenerative disease, comprising identifying a subject whohas experienced progression of a cognitive or physical disability andinitiating treatment of the subject by a method as described herein. Incertain embodiments, the method further comprises testing the severityof the subject's cognitive or physical disability to determine a scorerepresentative of the state of the subject's cognitive or physicaldisability after receiving the treatment for at least about six months,and, optionally, comparing the score to a score determined for thesubject prior to or at about the time of initiating the treatment.

Another aspect of the invention relates to a method of terminatingestrogen therapy in a subject receiving both estrogen therapy andanother immunotherapeutic for the treatment of a neurodegenerativedisease, such as multiple sclerosis, wherein the estrogen therapycomprises administering an original dose of estrogen on a regular basis(e.g., daily), by administering to the subject about half of theoriginal dose of the estrogen on the regular basis for a first period of1-3 weeks (preferably about 2 weeks), then administering to the subject,for a second period of 1-3 weeks (preferably about 2 weeks), about onequarter of the original dose of the estrogen; and after the secondperiod, terminating administration of the estrogen to the patient. Incertain such embodiments, the estrogen therapy further comprisesadministering a progestrogen on an intermittent basis, and the methodfurther comprises terminating administration of the progestrogen to thepatient before the first period or during the first period, preferablybefore the first period. The estrogen and progestrogen can be anyestrogen or progestrogen described herein, and the original dose can beany suitable therapeutic dose described herein. Administering half orone quarter of the original dose may be accomplished by changing thefrequency of administration, the amount of the estrogen administered, orboth.

In other aspects, the invention relates to compounds for use in treatingneurodegenerative diseases according to any of the various methodsdisclosed herein, use of compounds in the manufacture of medicaments forcarrying out any of the various methods disclosed herein, and kitscomprising compounds together with instructions for administering thecompounds according to any of the various methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting annualized relapse rates with Copaxone®plus estriol treatment as compared to Copaxone® plus estriol placebotreatment.

FIG. 2 is a bar graph depicting Paced Serial Addition Test (PASAT)cognitive test scores for all subjects (All), subjects with baselinescores of less than 55/60 (<55), and subjects with baseline scoresgreater than or equal to 55/60 (≥55). Comparison is made betweentreatment groups receiving Copaxone® plus estriol or Copaxone® plusestriol placebo. Data are expressed as percent change from baseline ineach treatment group.

FIG. 3 is a graph depicting the proportion of all subjects who hadsustained improvement of 3 points in PASAT scores for 6 months.Comparison is made between treatment groups receiving Copaxone® plusestriol (upper curve) or Copaxone® plus estriol placebo (lower curve).

FIG. 4 is a graph depicting Expanded Disability Status Scale (EDSS)scores over 24 months for treatment groups receiving Copaxone® plusestriol (slope=−0.11, p=0.06) or Copaxone® plus estriol placebo(slope=−0.01, p=0.90).

FIG. 5A is a bar graph depicting the number of subjects with brain MRIsthat had enhancing lesions at 12 months in subjects receiving Copaxone®plus estriol. 0, 12, and 24 refer to months on treatment.

FIG. 5B is a bar graph depicting the number of subjects with brain MRIsthat had enhancing lesions at 12 months in subjects receiving Copaxone®plus estriol placebo. 0, 12, and 24 refer to months on treatment.

FIG. 6 is a bar graph depicting estriol blood levels induced bytreatment with estriol 8 mg per day every day plus norethindrone 0.7 mga day for 14 days every 3 months. Levels are expressed as ng/ml. Datafor “month 0” represents pre-treatment baseline.

DETAILED DESCRIPTION

Approximately 50% of people diagnosed with multiple sclerosis (MS) willdevelop problems with cognition. Currently, there are no FDA-approvedtreatments targeting cognitive function in MS. Multiple sclerosisrelapses am known to be significantly decreased by approximately 80%during late pregnancy. This disease improvement may be due to estriol,an estrogen unique to pregnancy. Estriol blood levels go fromundetectable levels prior to pregnancy, increase during pregnancy andreach highest levels during late pregnancy. Further, estrogen treatmenthas been shown to have favorable effects on cognition in animal modelsof other neurological diseases.

An aspect of the invention is a method of treating multiple sclerosis.The method includes the steps of administering to a subject in needthereof, on a continuous basis throughout two or more consecutivetreatment periods, a therapeutically effective amount of an estrogen;and administering to the subject, for only a portion of each treatmentperiod, a therapeutically effective amount of a progestrogen.

The term “estrogen” as used herein refers to any biologically activeform of estrogen or precursor thereof. The term “estrogen” thus embracesnaturally occurring, synthetic, and semi-synthetic forms of estrogen,and biologically active, pharmaceutically acceptable salts and estersthereof. In certain embodiments, estrogen is selected from estriol (E3),estradiol (E2), estrone (E1), an ester thereof, a pharmaceuticallyacceptable salt of an ester thereof, and any combination of theforegoing. In certain embodiments, estrogen is estriol (E3) or an esterthereof, or a pharmaceutically acceptable salt of an ester thereof. Forexample, the estrogen can be estriol, estriol succinate, estrioldihexanoate, or estriol sulfate. In other embodiments, estrogen isestradiol (E2) or an ester thereof, or a pharmaceutically acceptablesalt of an ester thereof, while in yet other embodiments, estrogen isestrone (E1) or an ester thereof, or a pharmaceutically acceptable saltof an ester thereof. In certain preferred embodiments, estrogen isestriol (E3). In certain embodiments, estrogen is estradiol (E2). Incertain embodiments, estrogen is estrone (E1).

In certain embodiments, the estrogen is administered in a dose equal orequivalent to about 200 μg to about 20 mg estriol daily. For example, adose of 2 to 4 mg of estriol is generally considered to be equivalent to0.6 to 1.25 mg of conjugated estrogens or estrone. In certainembodiments, the estrogen is administered in a dose equal or equivalentto about 1 mg to about 10 mg estriol daily, preferably equal orequivalent to about 8 mg estriol daily. In most preferred embodiments,the estrogen is estriol administered in a dose of about 8 mg estrioldaily.

In certain embodiments, the estrogen is formulated for oraladministration. e.g., in a dose equal or equivalent to about 200 μg toabout 20 mg estriol daily. For example, a dose of 2 to 4 mg of estriolis generally considered to be equivalent to 0.6 to 1.25 mg of conjugatedestrogens or estrone. In certain embodiments, the estrogen is formulatedfor oral administration in a dose equal or equivalent to about 1 mg toabout 10 mg estriol daily, preferably equal or equivalent to about 8 mgestriol daily. In most preferred embodiments, the estrogen is estriolformulated for oral administration in a dose of about 8 mg estrioldaily.

In certain embodiments, the estrogen is orally administered in a doseequal or equivalent to about 200 μg to about 20 mg estriol daily. Forexample, a dose of 2 to 4 mg of estriol is generally considered to beequivalent to 0.6 to 1.25 mg of conjugated estrogens or estrone. Incertain embodiments, the estrogen is orally administered in a dose equalor equivalent to about 1 mg to about 10 mg estriol daily, preferablyequal or equivalent to about 8 mg estriol daily. In most preferredembodiments, the estrogen is estriol orally administered in a dose ofabout 8 mg estriol daily.

An “effective amount”, as used herein, refers to an amount that issufficient to achieve a desired biological effect. A “therapeuticallyeffective amount”, as used herein refers to an amount that is sufficientto achieve a desired therapeutic effect. For example, a therapeuticallyeffective amount can refer to an amount that is sufficient to improve atleast one sign or symptom of MS.

A therapeutically effective dose of the estrogen is, in someembodiments, one sufficient to raise the serum concentration above basallevels, and preferably to pregnancy levels or above pregnancy levels. Incertain embodiments, the therapeutically effective dose of the estrogenis selected to result in serum levels in a patient equivalent to thesteroid hormone level of that agent in women in the second or thirdtrimester of pregnancy.

For example, during the normal female menstrual cycle estradiol levelsare in the range of about 350 pg/ml scrum. During pregnancy, there isabout a 100-fold increase in the level of estradiol to about 10,000 toabout 35,000 pg/ml serum. Correale et al., J Immunol 161:3365-74 (1998)and Gilmore et al., J Immunol 158:446-51 (1997). In contrast, estriollevels are undetectable during the menstrual cycle in the non-pregnantstate. Estradiol levels rise progressively during pregnancy to levelsfrom 3,000 to 30,000 pg/ml (3 to 30 ng/ml).

In one embodiment, where the estrogen is estriol, the dose is from about4 to 16 milligrams daily, and more specifically, about 8 milligramsdaily. In this embodiment, blood serum levels preferably reach at leastabout 2 ng/ml, may reach about 10 to about 35 so ag/m, or mostpreferably about 20-30 ng/m. Sicotte et al. Neurology 56:A75 (2001). Insome embodiments, estradiol (E2) levels would preferably reach at leastabout 2 ng/ml and most preferably about to 10-35 ng/ml. In someembodiments, estrone (E1) levels would preferably reach at least about 2ng/ml and most preferably about 5-18 ng/ml. DeGroot et al.,Endocrinology 3(9):2171-223 (1994).

The dosage of the estrogen may be selected for an individual patientdepending upon the route of administration, severity of disease, age andweight of the patient, other medications the patient is taking and otherfactors normally considered by the attending physician, when determiningthe individual regimen and dosage level as the most appropriate for aparticular patient. Furthermore, the exact individual dosages can beadjusted somewhat depending on a variety of factors, including thespecific combination of the agents being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the particular disease beingtreated, the severity of the disorder, and the anatomical location ofthe disorder. Some variations in the dosage can be expected. In vitro orin vivo assays can be employed to help identify optimal dosage ranges.

The therapeutically effective dose of the estrogen included in thedosage form is selected at least by considering the type of estrogenselected and the mode of administration. The dosage form may include theestrogen in combination with other inert ingredients, includingadjuvants and pharmaceutically acceptable carriers for the facilitationof dosage to the patient as known to those skilled in the pharmaceuticalarts. The dosage form may be any form suitable to cause de estrogen toenter into the tissues of the patient.

Pharmaceutically acceptable carriers can optionally comprise a suitableamount of a pharmaceutically acceptable excipient so as to provide theform for proper administration. Pharmaceutical excipients can beliquids, such as water and oils, including those of petroleum, animal,vegetable, or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. The pharmaceutical excipients can include,for example, saline, gum acacia, gelatin, starch paste, tale, keratin,colloidal silica, urea and the like. In addition, auxiliary,stabilizing, thickening, lubricating, and coloring agents can be used.In one embodiment, the pharmaceutically acceptable excipients aresterile when administered to a subject. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In one embodiment, the dosage form of the estrogen is an oralpreparation (liquid, tablet, capsule, caplet, or the like) which whenconsumed results in elevated serum estrogen levels. The oral preparationmay comprise conventional carriers including diluents, binders,time-release agents, lubricants, and disintegrants.

In other embodiments of the invention, the dosage form of the estrogenmay be provided in a topical preparation (lotion, cream, ointment,patch, or the like) for transdermal application.

Alternatively, the dosage form may be provided as a suppository or thelike for transvaginal or transrectal application.

However, in other embodiments, the dosage form may also allow forpreparations to be applied subcutaneously, intravenously,intramuscularly, or via the respiratory system.

The term “progestrogen” (also known as “gestagen”) as used herein refersto any steroid hormone that binds to and activates a progesteronereceptor, or a precursor thereof. The term “progestrogen” thus embracesnaturally occurring, synthetic, and semi-synthetic forms ofprogestrogen, and biologically active, pharmaceutically acceptable saltsand esters thereof.

In certain embodiments, the progestrogen is selected from chlormadinoneacetate, cyproterone acetate, desogestrel, dienogest,5α-dihydroprogesterone, drospirenone (Yasmin®), ethinodiol acetate,ethynodiol diacetate, etonogestrel (Nexplanon®), gestodene,17-hydroxyprogesterone, levonorgestrel (Alesse®), medroxyprogesteroneacetate (17α-hydroxy-6α-methylprogesterone acetate; Provera®),megestrol, megestrol acetate(17α-acetoxy-6-dehydro-6-methylprogesterone), nestorone, nomegestrolacetate, norethindrone, norethindrone acetate (also known asnorethisterone acetate), norethynodrel (Enovid®), norgestimate,norgestrel, progesterone, tanaproget, trimegestone, or apharmaceutically acceptable salt of any of the foregoing, or anycombination thereof.

In certain embodiments, progestrogen is a progestin. The term“progestin” as used herein refers to a synthetic progestrogen as definedherein. Examples of progestins include desogestrel, dienogest,drospirenone (Yasmin®), ethinodiol acetate, etonogestrel (Nexplanon®),gestodene, levonorgestrel (Alesse®), medroxyprogesterone acetate(Provera®), nestorone, nomegestrol acetate, norethindrone, norethindroneacetate, norethynodrel (Enovid®), norgestimate, norgestrel, andtrimegestone.

In certain embodiments, the progestrogen is selected from progesterone,17-hydroxyprogesterone, 5α-dihydroprogesterone, norethindrone,norethindrone acetate (also known as norethisterone acetate),medroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone acetate),megestrol acetate (17α-acetoxy-6-dehydro-6-methylprogesterone),desogestrel, levonorgestrel, chlormadinone acetate, and cyproteroneacetate, pharmaceutically acceptable salts of any of the foregoing, andany combination thereof. In certain embodiments, progestrogen isselected from progesterone, 17-hydroxyprogesterone,5α-dihydroprogesterone, norethindrone, norethindrone acetate (also knownas norethisterone acetate), desogestrel, levonorgestrel, chlormadinoneacetate, and cyproterone acetate, pharmaceutically acceptable salts andesters of any of the foregoing, and any combination thereof. In certainembodiments, progestrogen is norethindrone or an ester thereof, or apharmaceutically acceptable salt of an ester thereof, preferablynorethindrone. In certain embodiments, progestrogen is progesterone oran ester thereof, or a pharmaceutically acceptable salt of an esterthereof.

In certain embodiments, the progestrogen is administered in a dose equalor equivalent to about 70 μg to about 7 mg norethindrone daily, such asabout 1 μg to about 1 mg norethindrone daily, most preferably in a doseequal or equivalent to about 0.7 mg norethindrone daily. In certainpreferred embodiments, the progestrogen is norethindrone administered ina dose of 0.7 mg norethindrone daily.

In certain embodiments, the progestrogen is formulated for oraladministration, e.g., in a dose equal or equivalent to about 70 μg toabout 7 mg norethindrone daily, such as about 100 μg to about 1 mgnorethindrone daily, most preferably in a dose equal or equivalent toabout 0.7 mg norethindrone daily. In certain preferred embodiments, theprogestrogen is norethindrone formulated for oral administration in adose of 0.7 mg norethindrone daily.

In certain embodiments, the progestrogen is orally administered in adose equal or equivalent to about 70 μg to about 7 mg norethindronedaily, such as about 100 μg to about 1 mg norethindrone daily, mostpreferably in a dose equal or equivalent to about 0.7 mg (i.e., 700 μg)norethindrone daily. In certain preferred embodiments, the progestrogenis norethindrone orally administered in a dose of 0.7 mg (i.e., 700 μg)norethindrone daily.

The therapeutically effective dose of the progestrogen included in thedosage form can be selected at least by considering the type ofprogestrogen selected and the mode of administration. The dosage formmay include the progestrogen in combination with other inertingredients, including adjuvants and pharmaceutically acceptablecarriers for the facilitation of dosage to the patient as known to thoseskilled in the pharmaceutical arts. The dosage form may be any formsuitable to cause the progestrogen to enter into the tissues of thepatient.

In one embodiment, the dosage form of the progestrogen is an oralpreparation (liquid, tablet, capsule, caplet, or the like) which whenconsumed results in elevated scrum progestogen levels. The oralpreparation may comprise conventional carriers including diluents,binders, time-release agents, lubricants, and disintegrants.

In other embodiments of the invention, the dosage form of theprogestrogen may be provided in a topical preparation (lotion, cream,ointment, patch, or the like) for transdermal application.

Alternatively, the dosage form may be provided as a suppository or thelike for transvaginal or transrectal application.

The estrogen is administered to the subject on a continuous basisthroughout two or more consecutive treatment periods. In certainembodiments a continuous basis means daily, i.e., on consecutive days.For example, estrogen administered orally to a subject on a daily basisthroughout two or more consecutive treatment periods is deemed to beestrogen administered to the subject on a continuous basis throughouttwo or more consecutive treatment periods. Alternatively, estrogenadministered transdermally to a subject on a daily basis throughout twoor more consecutive treatment periods is deemed to be estrogenadministered to the subject on a continuous basis throughout two or moreconsecutive treatment periods.

As used herein, a “treatment period” refers to a period of time duringwhich a subject is receiving, on a continuous or daily basis, at leastone therapeutic agent administered for the purpose of treating MS in thesubject. In certain embodiments, each treatment period is at least 28consecutive days. In certain embodiments, each treatment period is atleast 56 consecutive days. In certain embodiments, each treatment periodis at least 84 consecutive days. In certain embodiments, each treatmentperiod is at least 112 consecutive days. In certain embodiments, eachtreatment period is at least 140 consecutive days. In certainembodiments, each treatment period is at least 168 consecutive days.

In certain embodiments, each treatment period is at least 4 consecutiveweeks. In certain embodiments, each treatment period is at least Kconsecutive weeks. In certain embodiments, each treatment period is atleast 12 consecutive weeks. In certain embodiments, each treatmentperiod is at least 16 consecutive weeks. In certain embodiments, eachtreatment period is at least 20 consecutive weeks. In certainembodiments, each treatment period is at least 24 consecutive weeks.

In certain embodiments, each treatment period is at least one month. Incertain embodiments, each treatment period is at least two consecutivemonths. In certain embodiments, each treatment period is at least threeconsecutive months. In certain embodiments, each treatment period is atleast four consecutive months. In certain embodiments, each treatmentperiod is at least five consecutive months. In certain embodiments, eachtreatment period is at least six consecutive months.

The progestrogen is administered to the subject for only a portion ofeach treatment period. As used herein, “for only a portion of eachtreatment period” refers generally to a period of time that occursduring but is at least one day shorter than a treatment period. In apreferred embodiment, the phrase “for only a portion of each treatmentperiod” refers generally to a period of consecutive days that occursduring but is at least one day shorter than a treatment period.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 7 consecutive days of each treatment period. Forexample, if the treatment period is 28 days, in various embodiments theportion of such treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive days. Forconvenience, such portion can begin on day 1 of a treatment period, suchthat, for this example, the portion can encompass day 1 or days to 2, to3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1to 20, or 1 to 21.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 14 consecutive days of each treatment period. Forexample, if the treatment period is 28 days, in various embodiments theportion of such treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 consecutive days. For convenience, such portion canbegin on day 1 of a treatment period, such that, for this example, theportion can encompass day 1 or days 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or 1 to14.

In certain embodiments, the portion of each treatment period is dailyfor up to 7 consecutive days of each treatment period. For example, ifthe treatment period is 28 days, in various embodiments the portion ofsuch treatment period can be 1, 2, 3, 4, 5, 6, or 7 consecutive days.For convenience, such portion can begin on day 1 of a treatment period,such that, for this example, the portion can encompass day 1 or days 1to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7.

In certain embodiments, the portion of each treatment period is dailyfor up to 14 consecutive days of each treatment period. For example, ifthe treatment period is 28 days, in various embodiments the portion ofsuch treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 consecutive days. For convenience, such portion can begin on day 1of a treatment period, such that, for this example, the portion canencompass day 1 or days 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7,1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or 1 to 14.

In certain embodiments, the portion of each treatment period is dailyfor all but at least half of each treatment period. For example, if thetreatment period is 28 days, in various embodiments the portion of suchtreatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14consecutive days. For convenience, such portion can begin on day 1 of atreatment period, such that, for this example, the portion can encompassday 1 or days 1 to 2, to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or 1 to 14.

Preferably the progestrogen is administered to the subject for only aportion of each treatment period. During the remainder of the treatmentperiod, in certain embodiments the subject can receive estrogen butneither progestrogen nor a placebo in place of the progestrogen.Alternatively, during the remainder of the treatment period, in certainembodiments the subject can receive both estrogen and a placebo in placeof the progestrogen.

An aspect of the invention is a method of treating multiple sclerosis.The method includes the steps of administering orally to a subject inneed thereof, on a continuous basis for 84 consecutive days (12 weeks),about 8 mg of estriol daily: and administering orally to the subject,for 14 consecutive days (2 weeks) of the 84 consecutive days (12 weeks),about 0.7 mg of progestrogen daily. In certain embodiments, the 14consecutive days (2 weeks) are the first 14 consecutive days (2 weeks)of the 84 consecutive days (12 weeks). That is, if the 84 consecutivedays of estrogen administration are deemed to start on day 1, theprogestrogen is administered on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and 14, and then stopped. In certain embodiments, the subjectmay then continue to receive estrogen but neither progestrogen nor aplacebo in place of the progestrogen for the remaining 70 days. In otherembodiments, the method further includes the step of administering tothe subject a placebo in place of the progestrogen on each of the daysthe progestrogen is not administered to the subject. That is, thesubject may then receive both estrogen and a placebo in place of theprogestrogen for the remaining 70 days.

An aspect of the invention is a method of treating multiple sclerosis.The method includes the steps of administering orally to a subject inneed thereof, on a continuous basis for 84 consecutive days (12 weeks),about 8 mg of estriol daily; and administering orally to the subject,for 14 consecutive days (2 weeks) of the 84 consecutive days (12 weeks),about 0.7 mg of norethindrone daily. In certain embodiments, the 14consecutive days (2 weeks) are the first 14 consecutive days (2 weeks)of the 84 consecutive days (12 weeks). That is, if the 84 consecutivedays of estrogen administration are deemed to start on day 1, thenorethindrone is administered on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and 14, and then stopped. In certain embodiments, the subjectmay then continue to receive estrogen but neither norethindrone nor aplacebo in place of the norethindrone for the remaining 70 days. Inother embodiments, the method further includes the step of administeringto the subject a placebo in place of the norethindrone on each of thedays the norethindrone is not administered to the subject. That is, thesubject may then receive both estrogen and a placebo in place of thenorethindrone for the remaining 70 days.

The term “subject” as used herein refers to a living mammal and may beinterchangeably used with the term “patient”. In certain embodiments,the subject is a human. Preferably, the human subject is female, such asa woman. In certain embodiments, die subject is a premenopausal orperimenopausal woman. In certain embodiments, the subject is apremenopausal woman. In certain embodiments, the subject is aperimenopausal woman. In certain embodiments, the subject is apostmenopausal woman.

In certain embodiments, the multiple sclerosis is relapsing-remittingmultiple sclerosis. In certain embodiments, the multiple sclerosis issecondary-progressive multiple sclerosis. In certain embodiments, themultiple sclerosis is primary-progressive multiple sclerosis. In certainembodiments, the multiple sclerosis is progressive-relapsing multiplesclerosis. In certain embodiments, the subject has a mild form of anyone of the foregoing subtypes of MS. In certain embodiments, the subjecthas a moderate form of any one of the foregoing subtypes of MS. Incertain embodiments, the subject has an aggressive form of any one ofthe foregoing subtypes of MS.

In certain embodiments, the multiple sclerosis is, more accurately,so-called clinically isolated syndrome (CIS). Estriol can be used, inaccordance with the invention, to prevent or delay the onset ofrelapsing-remitting MS in subjects having CIS.

The various methods disclosed herein can be methods for improvingwalking, vision, balance, cognition, or other symptoms in a subject,such as a subject with multiple sclerosis, and/or methods for improvingmultiple sclerosis functional composite (MSFC), EDSS, or MSSS scores ina subject, such as a subject with multiple sclerosis. Thus, in certainembodiments, the methods of treatment disclosed herein include methodsfor stabilizing or improving disability in a patient, whereby thepatient's disability score (as measured by either of these tests oranother suitable test) after six months, one year, or two years oftherapy is at least about 10%, at least about 25%, at least about 40%,at least about 50%, or even at least about 60% higher relative to acontrol patient not receiving the estrogen/progestrogen therapy (butotherwise receiving the same treatment as the estrogen-treated patient).Alternatively, the patient's disability score (as measured by either ofthese tests or another suitable test) after six months, one year, or twoyears of therapy is within about 2% or within about 5% of an earlierassessment, or at least about 2%, at least about 5%, at least about atleast about 10%, at least about 25%, at least about 40%, at least about50%, or even at least about 60% higher than the earlier assessment.

For example, progression of a walking disability can be tested using awalking test, e.g., assessing the subject's performance on a 25-footwalk test at different points in time, such as at 0 months (baseline), 6months, 1 year, and 2 years. In certain embodiments, if there isdocumented worsening in walking (takes more seconds) by 20 percent ascompared to baseline (optionally if this worsening is confirmed on asubsequent walk test (e.g., 3 months later)), then the subject is deemedto have progressive worsening in walking. For such a patient not alreadyreceiving estrogen/progestrogen therapy, the subject demonstrating theprogressive walking disability commences treatment with estrogen, e.g.,estriol. The walking test may be repeated (e.g., at 1 year and/or 2years from the start of estrogen treatment) to assess whether theestrogen treatment slowed or halted any further worsening in walkingperformance, e.g., as measured by the walking test.

Improvements in cognition outcomes associated with MS therapy, whetherslowing of cognitive decline, stabilization of cognitive decline, orimprovement of cognitive function, can be assessed using the PASAT(e.g., PASAT 2 or PASAT 3) or SDMT test, or alternatively the MS-COGtest (see Erlanger et al., J Neuro Sci 340: 123-129 (2014)). Thus, incertain embodiments, the methods of treatment disclosed herein includemethods for stabilizing or improving cognition in a patient, whereby thepatient's cognition outcome after one year of therapy is at least about2%, at least about 5%, at least about 10%, at least about 25%, at leastabout 40%, at least about 50%, or even at least about 60% higherrelative to a control patient not receiving the estrogen/progestogentherapy (but otherwise receiving the same treatment as theestrogen-treated patient). e.g., as measured by any of the precedingtests. Alternatively, the patient's cognition outcome after six months,one year, or two years of therapy may be within about 2% or within about5% of an earlier assessment, or at least about 2%, at least about 5%, atleast about 10%, at least about 25%, at least about 40%, at least about50%, or even at least about 60% higher than the earlier assessment,e.g., as measured by any of the preceding tests at different times.

For example, a subject who scores below 50 on PASAT (and optionally ifsuch low 13 score is verified upon a second subsequent test, such aswithin one week to one month of the first) may be deemed to havecognitive disability. For such a patient not already receivingestrogen/progestrogen therapy, the subject demonstrating the cognitivedisability may commence treatment with estrogen. e.g., estriol. Incertain embodiments, the cognitive test may be repeated (e.g., at aboutsix months from the start of estrogen treatment) to assess whether theestrogen treatment slowed or halted any further worsening in cognitiveperformance, e.g., as measured by the PASAT test. In certain suchembodiments, the patient's score may increase by at least 3 points overthe course of six to twelve months of estrogen therapy.

While the various methods disclosed herein are typically efficaciouswhen administered without additional therapeutics, in certainembodiments, any of these methods further includes the step ofadministering to the subject an immunotherapeutic agent, wherein theimmunotherapeutic agent is neither an estrogen nor a progestrogen. Thatis, in certain embodiments the subject is administered, in addition tothe estrogen and progestrogen (or placebo), a third agent useful in thetreatment of MS. Such agents useful in the treatment of MS are, ingeneral, immunotherapeutic agents. At least in connection with MS, suchagents are sometimes referred to as disease-modifying therapies ordisease-modifying therapeutics (DMTs).

The term “immunotherapeutic agent” as used herein refers to a compoundwith an objectively measurable effect on at least one aspect of theimmune system or an immune response. In certain embodiments, theimmunotherapeutic agent is immunosuppressive, i.e., it exerts anobjectively measurable inhibitory effect on at least one aspect of theimmune system or an immune response. In certain embodiments, theimmunotherapeutic agent is anti-inflammatory. In certain embodiments,the immunotherapeutic agent is a small molecule (molecular weight lessthan or equal to about 1.5 kDa) pharmaceutical compound or composition.In certain embodiments, the immunotherapeutic agent is a biologicalcompound or composition, e.g., an antibody, peptide, nucleic acid, etc.

In certain embodiments, the immunotherapeutic agent is not an estrogen.In certain embodiments, the immunotherapeutic agent is not aprogestrogen. Preferably, the immunotherapeutic agent is neither anestrogen nor a progestrogen.

In certain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Teefidera®; BG-12, which may be administered in anamount from about 220 mg to about 260 mg per day, such as about 220 mg,240 mg, or 260 mg per day), fingolimod (Gilenya®, which may beadministered in an amount from about 0.25 mg to about 0.75 mg per day,such as about 0.25 mg, 0.50 mg, or 0.75 mg per day), glatiramer acetate(Copaxone®, for example “longer-lasting” 40 mg/mi or 20 mg/m versions),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (Betaseron®or Extavia®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), andteriflunomide (Aubagio®, which may be administered in an amount fromabout 7 mg to about 14 mg per day, such as about 7 mg, 10 mg, or 14 mgper day), mycophenolate mofetil, paclitaxel, cyclosporine,corticosteroids (e.g., prednisone, methylprednisolone), azathioprine,cyclophosphamide, methotrexate, cladribine, 4-aminopyridine, andtizanidine. In certain embodiments, the immunotherapeutic agent isselected from dimethyl fumarate (Teefidera®; BG-12), fingolimod(Gilenya®), glatiramer acetate (Copaxone®), interferon beta-1a (Avonex®or Rebif®), interferon beta-1b (Betaseron® or Extavia®), mitoxantrone(Novantrone®), natalizumab (Tysabri®), and teriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is dimethyl fumarate(Teefidera®; BG-12). In certain embodiments, the immunotherapeutic agentis fingolimod (Gilenya®). In certain embodiments, the immunotherapeuticagent is glatiramer acetate (Copaxone®). In certain embodiments, theimmunotherapeutic agent is interferon beta-1a (Avonex® or Rebif®). Incertain embodiments, the immunotherapeutic agent is interferon beta-1b(Betaseron® or Extavia®). In certain embodiments, the immunotherapeuticagent is mitoxantrone (Novantrone®). In certain embodiments, theimmunotherapeutic agent is natalizumab (Tysabri®). In certainembodiments, the immunotherapeutic agent is teriflunomide (Aubagio®).

In certain embodiments, the subject is already receiving adisease-modifying therapeutic. In this circumstance, the subject cancontinue to receive the disease-modifying therapeutic while taking theestrogen, with and without the progestogen. Significantly, however, thedose of the disease-modifying therapeutic may be decreased when used incombination with the estrogen, with and without the progestrogen. Forexample, a current standard dose for glatiramer acetate (Copaxone®) is40 mg subcutaneously (s.c.) three times a week, or 20 mg s.c. daily. Inconjunction with estrogen and progestogen in accordance with theinvention, the dose for glatiramer acetate (Copaxone®) may be reduced byup to 50 percent or more, e.g., to 20 mg s.c. three times a week.

As another example, a current standard dose for fingolimod (Gilenya®) is0.5 mg by mouth (p.o.) daily. In conjunction with estrogen andprogestrogen in accordance with the invention, the dose for fingolimod(Gilenya®) may be reduced by up to 50 percent or more, e.g., to 0.25 mgp.o. daily.

As another example, a current standard dose for dimethyl fumarate(Teefidera®) is 240 mg p.o. daily. In conjunction with estrogen andprogestrogen in accordance with the invention, the dose for dimethylfumarate (Teefidera®) may be reduced by up to S) percent or more. e.g.,to 120 mg p.o. daily.

As yet another example, a current standard dose for interferon beta-1a(Avonex® or Rebif®) is 30 μg intramuscularly (i.m.) weekly (Avonex®) or44 μg s.c. three days a week (Rebif®). In conjunction with estrogen andprogestrogen in accordance with the invention, Z5 the dose for Avonex®may be reduced to 15 μg i.m. weekly, and the dose for Rebif® may bereduced to 22 μg s.c. three days a week.

As yet another example, a current standard dose for interferon beta-1b(Betaseron® or Extavia®) is 0.25 mg s.c. every other day (Betaseron® orExtavia®). In conjunction with estrogen and progestrogen in accordancewith the invention, the dose for interferon beta-1b (Betaseron® orExtavia®) may be reduced to 0.125 mg s.c. every other day.

In certain embodiments, the subject is receiving an immunotherapeuticagent and has cognitive disability. For example, if a subject scoresbelow 50 on PASAT, and optionally if such low score is verified uponretest within about one week to one month, then the subject may bedeemed to have cognitive disability. In accordance with the invention,this cognitive disability is treated with estrogen, e.g., estriol, and,in certain embodiments, followed up with further retest e.g., about sixmonths from the start of estrogen treatment, such as to achieve anincrease in test score of at least 3 points.

In certain embodiments, the subject is receiving an immunotherapeuticagent and has progressive walking disability. For example, the subjectperforms a 25 foot walk test, e.g., at 0 months (baseline), 6 months, 1year, and/or 2 years. If there is documented worsening in walking (takesmore seconds), e.g., by 20 percent as compared to baseline, and thisworsening is confirmed on a repeated walk test, e.g., about 3 monthslater, then the subject is deemed to have progressive worsening inwalking. In accordance with the invention, this progressive walkingdisability is treated with estrogen, e.g., estriol, and, in certainembodiments, followed up with repeat walking test, e.g., at about 1 yearor 2 years from the start of estrogen treatment, such as to stabilize orhalt any further worsening in walking times.

In certain embodiments, the subject is receiving an immunotherapeuticagent and experiencing a relapse or progression of the multiplesclerosis. For example, a subject may experience a relapse orprogression while on a maintenance dose of a DMT. Such subject can thenbegin concurrent treatment with estrogen in accordance with any of thevarious methods disclosed herein, e.g., to reduce the frequency and/orseverity of relapses or to slow progression of the disease (e.g., asdetermined by assessment of one or more of walking, vision, balance,cognition, or other symptoms of the condition, e.g., as measuredaccording to the Expanded Disability Severity Scale (EDSS) and/or themultiple sclerosis functional composite (MSFC)). Thus, the variousembodiments of the methods disclosed herein can be methods for improvingwalking, vision, balance, cognition, or other symptoms in a subject,such as a subject with multiple sclerosis, and/or methods for improvingEDSS or MSFC scores in a subject, such as a subject with multiplesclerosis.

In certain embodiments, the subject is receiving an immunotherapeuticagent and experiencing a relapse of the multiple sclerosis. For example,a subject may experience a relapse while on a maintenance dose of a DMT.Such subject can then begin concurrent treatment with estrogen inaccordance with a method of the present invention, e.g., to reduce thefrequency and/or severity of relapses.

In certain embodiments, the subject is receiving an immunotherapeuticagent selected from interferon-beta 1a, interferon-beta 1b, glatirameracetate, natalizumab, mitoxantrone, fingolimod, teriflunomide, anddimethyl fumarate during a ramp-up period for dose of theimmunotherapeutic agent, e.g., the patient begins receiving theimmunotherapeutic and the estrogen therapy at the same time or at aboutthe same time (such as for patients who have not previously receivedtreatments for their disease). Advantageously, estrogen induces a rapidonset of therapeutic effect on MS, while commonly an immunotherapeuticagent such as interferon-beta 1a, interferon-beta 1b, glatirameracetate, natalizumab, mitoxantrone, fingolimod, teriflunomide, ordimethyl fumarate may take weeks to months to induce observableimprovements on some or all symptoms.

In certain embodiments, the subject is receiving glatiramer acetateduring a ramp-up period for dose of the glatiramer acetate. In othercertain embodiments, the subject is not already receiving adisease-modifying therapeutic.

In certain embodiments, the estrogen and the progestrogen are formulatedseparately from one another, e.g., the subject receives the estrogen asa single formulation and the progestrogen as a separate formulation. Fororal administration, a given dose of each formulation can comprise oneor more pills, tablets, capsules, or the like (i.e., unit doses). Forexample, an 8 mg dose of estriol can be administered as four 2 mgcapsules, and a 0.7 mg dose of norethindrone can be administered as asingle capsule, though preferably each dose is administered in a singleunit dose (e.g., one unit dose each for the estrogen and theprogestrogen).

In certain embodiments, e.g., where a placebo is administered with theestrogen on days when progestrogen is not administered, the estrogen andthe placebo are formulated separately from one another. For example, thesubject is administered the estrogen as a single formulation and theplacebo as a separate formulation. For oral administration, a given doseof each formulation can comprise one or more pills, tablets, capsules,or the like (i.e., unit doses). For example, an 8 mg dose of estriol canbe administered as four 2 mg capsules, and a placebo can be administeredas a single capsule.

When a given dose of any agent involves administration of more than asingle unit dose, e.g., four 2 mg capsules of estriol, the individualunit doses can be administered at essentially the same time, or they canbe administered at different times on a given day, provided the entiredaily dose is administered within a single day. For example, four 2 mgcapsules of estriol can be taken together essentially once a day, orthey may be taken two at a time twice a day, or they may be taken one ata time four times a day. Additional schedules are contemplated by theinvention, again provided the entire daily dose is administered within asingle day. While it may be preferable that the subject follow the sameschedule from one day to the next, such is not required once againprovided the entire daily dose is administered within a single day.

When the estrogen and the progestogen are formulated separately, theycan be administered essentially simultaneously, or they can beadministered sequentially with respect to each other. For example, inone embodiment the subject is administered four 2 mg capsules of estrioland one 0.7 mg capsule of norethindrone essentially simultaneously. Inanother embodiment, the subject is administered estriol in divideddoses, e.g., two 2 mg capsules twice daily, and the progestrogen isadministered essentially simultaneously with one of the divided doses ofestriol. In yet another embodiment, the subject is administered estriolin divided doses, e.g., two 2 mg capsules twice daily, and theprogestrogen is administered at a separate time from either one of thedivided doses of estriol.

Similarly, when the estrogen and the placebo are formulated separately,they can be administered essentially simultaneously, or they can beadministered sequentially with respect to each other. For example, inone embodiment the subject is administered four 2 mg capsules of estrioland one placebo essentially simultaneously. In another embodiment, thesubject is administered estriol in divided doses, e.g., two 2 mgcapsules twice daily, and the placebo is administered essentiallysimultaneously with one of the divided doses of estriol. In yet anotherembodiment, the subject is administered estriol in divided doses, e.g.,two 2 mg capsules twice daily, and the placebo is administered at aseparate time from either one of the divided doses of estriol.

In certain embodiments, the estrogen and the progestrogen are formulatedtogether. For oral administration, a given dose of each component,formulated together, can comprise one or more pills, tablets, capsules,or the like (i.e., unit doses). For example, an 8 mg dose of estriol anda 0.7 mg dose of norethindrone can be coformulated and administered asfour capsules, each containing 2 mg estriol and 0.0875 mg norethindrone,though preferably, where applicable, they are coformulated as one unitdose comprising both the estrogen and the progestrogen.

In certain embodiments, e.g., where a placebo is administered with theestrogen on days when progestrogen is not administered, the estrogen andthe placebo are formulated together. For oral administration, a givendose of each component, formulated together, can comprise one or morepills, tablets, capsules, or the like (i.e., unit doses). For example,an 8 mg dose of estriol and a placebo can be coformulated andadministered as four capsules, each containing 2 mg estriol and asuitable amount of placebo.

When a given dose of any coformulation of estriol and progestrogen (orplacebo) involves administration of more than a single unit dose, e.g.,four capsules, each containing 2 mg estriol and 0.0875 mg norethindrone,the individual unit doses can be administered at essentially the sametime, or they can be administered at different times on a given day,provided the entire daily dose is administered within a single day. Forexample, four capsules, each containing estriol and progestrogen (orplacebo) can be taken together essentially once a day, or they may betaken two at a time twice a day, or they may be taken one at a time fourtimes a day. Additional schedules are contemplated by the invention,again provided the entire daily dose is administered within a singleday. While it may be preferable that the subject follow the sameschedule from one day to the next, such is not required, once againprovided the entire daily dose is administered within a single day.

Clinically, MS can be assessed and monitored using any of a number ofstructural (anatomical) and functional tests, including, withoutlimitation: magnetic resonance imaging (MRI); Paced Serial Addition Test(PASAT); symbol digit modalities test (SDMT); expanded disability statusscore (EDSS); multiple sclerosis functional composite (MSFC); 25-footwalk test; 9-hole peg test; low contrast visual acuity; MS Quality ofLife; Modified Fatigue Impact Scale; Beck Depression Inventory; 7/24Spatial Recall Test; Benton Forms F & G; Buschke Selective RemindingTest; Verbal Paired Associates; Word List Generation. Recently, thePASAT test of cognitive function has come under criticism by some forits test-retest reliability and practice effect whereby one naturallyimproves over time with repeated test taking. Polman C H et al.,Neurology 74 Suppl 3: S8-15 (200). In some embodiments, assessment ofMacDonald dissemination in space and time finds use in the presentmethods. For example, for dissemination in space, lesion imaging, suchas, by way of illustration, Barkhof-Tintore MR imaging criteria, may beused. For instance, the following criteria can be evaluated: (1) atleast one gadolinium-enhancing lesion or 9 T2 hyperintense lesions; (2)at least one infratentorial lesion; (3) at least one juxtacorticallesion; (4) at least 3 periventricular lesions; and (5) a spinal cordlesion. Such imaging criteria can optionally be used in combination withevaluation for immunoglobulin abnormalities in the cerebrospinal fluid(CSF), for example. For dissemination in time, MR imaging can also beused. For example, if an MR imaging scan of the brain performed at ≥3months after an initial clinical event demonstrates a newgadolinium-enhancing lesion, this may indicate a new CNS inflammatoryevent, because the duration of gadolinium enhancement in MS is usuallyless than 6 weeks. If there am no gadolinium-enhancing lesions but a newT2 lesion (presuming an MR imaging at the time of the initial event), arepeat MR imaging scan after another 3 months may be needed withdemonstration of a new T2 lesion or gadolinium-enhancing lesion. Invarious embodiments, any one or more of these structural (anatomical)and functional tests may be used in conjunction with the presentinvention (e.g., to assess the effectiveness of a disclosed treatmentmethod).

A randomized, double-blind, placebo-controlled clinical trial wasdesigned to ascertain whether, in women, treatment with an estrogen pill(estriol), used in combination with major FDA-approved standardtreatments for MS (Betaseron®, Extavia®, Rebif®, Avonex®, Copaxone®,Gilenya®, Aubagio®, or Teefidera®) for one year, can improve cognitivetesting as compared to treatment with a placebo pill in combination withthe same major FDA-approved standard treatments for MS. Interim resultsfrom this study are presented in the Examples below.

With respect to the estriol intervention, the study design includescontinuous treatment with estriol, part of the time with norethindrone,and part of the time without norethindrone. That is, again with respectto the estriol intervention, the study can be understood as a series ofconsecutive periods, wherein for each period the subject continuouslyreceives estriol and, for only a portion of each period, the subjectalso receives norethindrone.

In the experimental group, subjects receive standard MS treatment plusestriol 8 ng by mouth daily (continuously) plus norethindrone 0.7 mg bymouth daily for two weeks starting at month 6 and at months 9 and 12.

In the control group, subjects receive standard MS treatment plusestriol placebo by mouth daily (continuously) plus norethindrone placeboby mouth daily for two weeks starting at month 6 and at months 9 and 12.

Study subjects are 18- to 50-year-old women with diagnosis of clinicallydefinite or MacDonald criteria relapsing-remitting MS,secondary-progressive MS, or primary-progressive MS; on a stable dos ofCopaxone®, Betaseron®, Extavia®, Rebif®, Avonex®, Gilenya®, Aubagio®, orTeefidera® for a minimum of 3 months duration prior to enrollment; andwith no relapse within 30 days before trial enrollment. Excluded fromthe study are women on oral contraceptives (OCP), hormone replacementtherapy (HRT), progesterone intrauterine devices (IUDs), or other sexhormones.

The primary outcome measure for this study is change from baseline incognitive function (processing speed), assessed by Paced Serial AdditionTest (PASAT). Numerical test scores (ranging from 0-60) are acquired,then percent change for each subject at trial conclusion as compared tobaseline is determined. A primary goal is to determine whether greaterimprovement as expressed as percent change occurs in the estriol groupas compared to the placebo group.

Secondary outcome measures include change from baseline in cognitivefunction as assessed by cognitive evoked potentials, measured inmilliseconds; change from baseline in standard MS outcome measures(relapses, expanded disability status score (EDSS), 25-foot walk test,9-hole peg test, low contrast visual acuity, MS Quality of Life,Modified Fatigue Impact Scale, and Beck Depression Inventory); changefrom baseline in cognitive function as assessed by a brief battery ofcognitive tests; and safety.

Cognitive evoked potentials are recorded in msecs for each subject atbaseline and conclusion. The percent improvement at conclusion ascompared to baseline for each subject is determined. Group comparisonswill reveal whether the percent improvement is greater in the estrioltreated group as compared to the placebo treated group.

A brief battery of cognitive tests is administered, including:Processing speed: symbol digit modalities test (SDMT); Visual memory:7/24 Spatial Recall Test, Benton Forms F & G; Verbal memory: BuschkeSelective Reminding Test, Verbal Paired Associates; and Language: WordList Generation. Each subject is tested at baseline, month 6, andconclusion. Percent change at conclusion as compared to baseline isdetermined in each subject. Group comparisons will reveal whichcognitive test within the battery had greater improvement in the estrioltreated group as compared to the placebo treated group.

Safety is measured based on neurologic exams, laboratory tests(chemistries, complete blood count (CBC)), and breast and gynecologicexams.

In some aspects, the invention relates to a method of slowing, halting,or reversing physical disability or stabilizing or improving physicaldisability in a female subject who has multiple sclerosis, comprisingadministering to the subject an estrogen and a secondary agent. Theestrogen may be administered orally. The estrogen may be formulated as apill, e.g., for oral administration. The estrogen may be administeredorally in a dose equal or equivalent to about 8 mg of estriol per day.The estrogen may be estriol. Estriol may be administered at a dosesufficient to induce an estriol level in the blood that is consistentwith a level observed during mid-pregnancy. Physical disability may beassessed, for example, using the expanded disability status scale(“EDSS”). The subject may have relapsing-remitting multiple sclerosis orsecondary progressive multiple sclerosis. For example, the subject mayhave secondary progressive multiple sclerosis and the estrogen may beadministered as a neuroprotective agent. The secondary agent may beglatiramer acetate copolymer 1. Glatiramer acetate copolymer 1 may beadministered, for example, by injection. The estrogen and secondaryagent may be administered for at least 12 months, such as for 12 months,13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, or 24 months. Theestrogen and secondary agent may be administered for at least 24 months.The method may further comprise evaluating the physical disability of asubject, e.g., by obtaining an EDSS score. The method may furthercomprise evaluating a change in the physical disability of a subject,e.g., by obtaining a first EDSS score, obtaining a second EDSS score,and comparing the first EDSS score and the second EDSS score, therebyevaluating the change in physical disability. The first EDSS score maybe obtained prior to administering the estrogen and the secondary agent,such as within a day, week, or month prior to administering the estrogenand the secondary agent. The second EDSS score may be obtained a periodof time after first administering the estrogen and the secondary agent,such as at least 12 months after first administering the estrogen andthe secondary agent, such as 12 months or 24 months after firstadministering the estrogen and the secondary agent.

In some embodiments, the invention relates to a method of slowing,halting, or reversing cognitive decline or stabilizing or improvingcognitive function in a female subject who has multiple sclerosis,comprising administering to the subject an estrogen and a secondaryagent. The estrogen may be administered orally. The estrogen may beformulated as a pill, e.g., for oral administration. The estrogen may beadminstered orally in a dose equal or equivalent to about 8 mg ofestriol per day. The estrogen may be estriol. Estriol may beadministered at a dose sufficient to induce an estriol level in theblood that is consistent with a level observed during mid-pregnancy.Cognitive function and or cognitive decline may be assessed, forexample, using the paced auditory serial additional test (“PASAT”). Thesubject may have relapsing-remitting multiple sclerosis or secondaryprogressive multiple sclerosis. For example, the subject may havesecondary progressive multiple sclerosis and the estrogen may beadministered as a neuroprotective agent. The secondary agent may beglatiramer acetate copolymer 1. Glatiramer acetate copolymer 1 may beadministered, for example, by injection. The estrogen and secondaryagent may be administered for at least 12 months, such as for 12 months,13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, or 24 months. Theestrogen and secondary agent may be administered for at least 24 months.The method may further comprise evaluating the cognitive function of asubject, e.g., by obtaining a PASAT score. The method may furthercomprise evaluating a change in the cognitive function of a subject,e.g., by obtaining a first PASAT score, obtaining a second PASAT score,and comparing the first PASAT score and the second PASAT score, therebyevaluating the change in cognitive function. The first PASAT score maybe obtained prior to administering the estrogen and the secondary agent,such as within a day, week, or month prior to administering the estrogenand the secondary agent. The second PASAT score may be obtained a periodof time after first administering the estrogen and the secondary agent,such as at least 12 months after first administering the estrogen andthe secondary agent, such as 12 months or 24 months after firstadministering the estrogen and the secondary agent.

In some embodiments, the invention relates to a method of treating ahuman patient exhibiting at least one clinical sign or symptom ofmultiple sclerosis (e.g., weakness, numbness, tingling, loss of vision,memory difficulty, extreme fatigue, gadolinium enhancing lesions, theaccumulation of T2 lesions, elevated Th1 cytokines (e.g., interferongamma), and/or reduced Th2 cytokines (e.g., IL-10), comprisingadministering to the patient an estriol, an immunomodulatory compound,and norethindrone. The method may ameliorate the at least one sign orsymptom of multiple sclerosis. The estriol may be selected from estriol,estriol succinate, estriol sulfamate, or estriol dihexanoate. Theestriol may be administered orally at a dose of about 1 mg to about 20mg per day, such as about 4 mg to about 16 mg. For example, the estriolmay be administered orally at a dose of 2 mg, 4 mg, 6 mg, 8 mg, 10 mg,12 mg, 14 mg, or 16 mg per day. Estriol may be administered at a dosesufficient to increase the scrum concentration of estriol in the patientto between about 2 ng/mL and about 30 ng/mL. The immunomodulatorycompound may be administered orally. The immunomodulatory compound maybe, for example, fingolimod, teriflunomide, dimethyl fumarate, or acombination thereof. For example, the immunomodulatory compound may befingolimod, and fingolimod may be administered at a dose of about 0.25mg to about 0.75 mg per day, such as about 0.25 mg, about 0.5 mg, orabout 0.75 mg per day. In some embodiments, fingolimod is administereddaily. The immunomodulatory compound may be teriflunomide, andteriflunomide may be administered at a dose of about 7 mg to about 14 mgper day, such as about 7 mg, about 10 mg, or about 14 mg per day. Insome embodiments, teriflunomide is administered daily. Theimmunomodulatory compound may be dimethyl fumarate, and dimethylfumarate may be administered at a dose of about 220 mg to about 260 mgper day, such as about 220 mg, about 240 mg, or about 260 mg per day. Insome embodiments, dimethyl fumarate is administered twice daily. Thenorethindrone may be administered orally at a dose of about 0.4 mg toabout 1.0 mg per day, e.g., for two weeks every three months. Forexample, the norethindrone may be administered orally at a dose of about0.7 mg per day, e.g., for two weeks every three months.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to limit the invention.

EXEMPLIFICATION Example 1—Use of Copaxone® and Estriol for the Treatmentof Multiple Sclerosis

This example describes a randomized, double-blind, placebo-controlledhuman clinical trial for the treatment of multiple sclerosis usingCopaxone® and estriol.

Enrollment Criteria

Eligible patients were female, between ages 18 and 50, had activerelapsing disease, and had an Expanded Disability Status Scale (EDSS)score between 0 and 4.5. Women who were pregnant, breastfeeding, takinghormone replacement therapy, or taking oral contraceptives were excludedfrom the trial.

Study Conduct and Monitoring Schedule

158 patients were randomized to Copaxone® (glatiramer acetate)injections (20 mg/day) and oral estriol (8 mg/day) or to Copaxone®injections and placebo for a 24-month treatment duration. Gynecologistsexamined the patients before, during, and after the study. Each patientwas examined at three- to six-month intervals during the trial. Patientsalso underwent mammograms before and after the study. In addition, atbaseline, three months, six months, 12 months, 18 months, and 24 months,the investigators measured participants' estriol levels, and assessedfor MS relapses and MS-related disabilities.

A total of 82 patients received Copaxone® plus estriol, and 76 patientsreceived Copaxone® plus placebo. Baseline characteristics were similarin both patient groups. Participants' mean age at entry wasapproximately 38, and their mean EDSS score at entry was 2.2. Estriollevels in serum were in a mid-pregnancy range in the estriol-treatedgroup. To ensure breast and uterus safety, every three months thepatients took norethindrone 0.7 mg once a day for 14 days. This hormoneregimen was found to be safe and well tolerated with regard to seriousadverse events, adverse events, general exams, blood chemistries, andhematological studies, as well as for gynecological outcomes (se Table1).

TABLE 1 Safety and Tolerability Data Breast - Uterine Fibrocysticendometrial breast Uterine thickness >8 Uterine disease on Patientfibroids on mm on endometrial clinical Group^(a) ultrasound ultrasoundbiopsies exam Mammograms Placebo 8 subjects 27 subjects 6 subjects 4subjects No breast with 41 with 10 cancer exams exams (no abnormalproliferation) Estriol 8 subjects 24 subjects 9 subjects 5 subjects with32 with 11 exams exams (no abnormal proliferation) ^(a)Patients receivedCopaxone ® with either estriol or placebo

Primary Outcome Measure

The primary outcome measure for disease efficacy was annualized relapserate. While most Phase II trials used surrogates or biomarkers as theprimary outcome, the trial focused on an outcome measure acceptable forapproval by the FDA. Since this was a Phase II trial, it was powered toreduce relapses by one third more in the Copaxone® plus estriol group ascompared to the Copaxone® plus placebo group, with a targeted p value ofp=0.10 at the end of study which was 24 months. As shown in FIG. 1,after 24 months of treatment the primary outcome measure was attained byreducing relapse rates by 32% (p=0.11) in the Copaxone® plus estriolgroup as compared to the Copaxone® plus placebo group. Surprisingly,after only 12 months of treatment, the relapse rate was reduced by 47%(p=0.02) in the Copaxone® plus estriol group as compared to theCopaxone® plus placebo group, see FIG. 1. Thus, in addition to findingthat Copaxone® plus estriol treatment had significant benefit inreducing the frequency of relapses over 24 months, the combinationtreatment also had a more rapid onset of action as compared to Copaxone®plus placebo.

These results were surprising given that estriol treatment was notcompared to a true placebo, but rather was tested in combination withstandard-of-car therapy (Copaxone®). Since anti-inflammatory drugs theFDA has approved have so far required much larger sample sizes to show asignificant reduction in relapse rates, even as compared to a trueplacebo, the results of the study adding estriol to Copaxone® suggest anovel mechanism of action, a mechanism never before observed in MS.

Cognitive Disability Assessment

The effect of treatment was assessed on cognitive disability with a testfor processing speed that has been extensively used in MS, the PacedAuditory Serial Addition Test (PASAT). A perfect score is 60, withscores lower than 55 indicating disability. By 12 months of treatment,scores on the PASAT improved by approximately 6% (i.e., 3 points),compared with scores at baseline, among patients receiving Copaxone®plus estriol (p=0.03). The change largely resulted from a 12%improvement (i.e., 6 points) among participants with cognitivedisability prior to treatment as reflected in scores of less than 55 atbaseline (of a maximum of 60), see FIG. 2. After 12 months of treatment,patients receiving Copaxone® plus estriol continued to have high PASATscores to the end of study at month 24, while participants receivingplacebo began to show improved PASAT scores by month 24. Notably, achange of six or more points in tests of processing speed in MS isconsidered to be clinically significant. Further, Copaxone® plus estrioltreatment improved function in those with significant cognitivedisability, rather than merely slowing cognitive decline. Thisrepresents repair of disability, not merely prevention of worsening.

Next it was shown that the improvement in PASAT cognitive test scoreswas sustained when subjects were followed for the entire 24 monthperiod, p=0.02 (FIG. 3).

In addition, the beneficial effects of estriol treatment on cognitivefunction were shown using another cognitive test, the 7/24 spatialrecall test (for spatial memory). While initial encoding of informationdid not differ between groups, the number of subjects with perfectscores for immediate recall (p=0.006) and delayed recall (p=0.04) washigher in the Copaxone® plus estriol treated group as compared to theCopaxone® plus placebo treated group over the entire 24 month treatmentduration. Such rapid and potent effects on cognition that were observedin the Copaxone® plus estriol group as compared to the Copaxone® plusplacebo group were surprising and point to a novel effect on cognitivedisability not seen before with other FDA-approved MS drugs.

Ambulatory Assessment

The Expanded Disability Status Scale (EDSS) is a standard compositedisability score used in MS trials. Higher scores indicate worsedisability. This composite covers a variety of disabilities (includingambulation, vision, cognition, coordination, etc.), but the scoring isnot linear and the composite score is understood to be principally anindicator of the level of disability in ambulation. While there was nochange in the EDSS scores for the Copaxone® plus placebo treatmentgroup, the Copaxone® plus estriol treatment group showed a significantdecrease (i.e., improvement) in this disability score (FIG. 4). Further,the probability of EDSS progression (as defined by an increase in EDSSof 1 point 13 for over 6 months) was 19% less likely in the Copaxone®plus estriol group, while the probability of EDSS improvement (asdefined by a decrease in EDSS of 1 point for over 6 months) was 23% morelikely in the Copaxone® plus estriol group.

Another clinical disability measure with treatment effects was the timed25-foot walk test. This test measures how many seconds it takes to walk25 feet, with higher scores indicating worse disability. The walk timewas significantly increased in the Copaxone® plus placebo group(p=0.03), while it was slightly decreased in the Copaxone® plus estriolgroup, together resulting in a significant between-group difference(p=0.02). Together these data show a gradual worsening in walking timesin the Copaxone® plus placebo treated group, which did not occur in theCopaxone® plus estriol treated group. This beneficial effect of estrioltreatment on 25-foot walking times is consistent with the beneficialeffect of estriol treatment on EDSS scores since the latter is weightedtoward being an indicator of ambulation.

Brain MRI is used extensively in MS as a surrogate marker for clinicaleffects. Brain white matter gadolinium enhancing lesions are a biomarkerfor relapses. It was found that the number of subjects with brain MRIscans that were positive for enhancing lesions was reduced by 56% atmonth 12 in the Copaxone® plus estriol treatment group vs. by 22% in theCopaxone®, plus placebo treatment group (p=0.14). This effect isconsistent with the observation that the Copaxone® plus estriol groupdemonstrated a more rapid onset of action in reducing relapse rates ascompared to the Copaxone® plus placebo group (FIG. 5A,B).

FIG. 6 depicts estriol blood levels induced by treatment with estriol 8mg per day every day plus norethindrone 0.7 mg a day for 14 days every 3months. Induced levels of about 10 ng/ml are comparable to the leveltypically observed in mid-pregnancy.

INCORPORATION BY REFERENCE

All patents, published patent applications, and other publicationsmentioned in the description above are incorporated by reference hereinin their entirety.

EQUIVALENTS

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

1-67. (canceled)
 68. A method of improving cognition in a subject withrelapsing-remitting multiple sclerosis, comprising administering orallyto a subject in need thereof, on a continuous basis for a treatmentperiod of 84 consecutive days (12 weeks), 8 mg of estriol daily; andadministering orally to the subject, for 14 consecutive days (2 weeks)of the treatment period, 0.7 mg of norethindrone daily; wherein themethod comprises at least four consecutive treatment periods; and thesubject is a woman.
 69. The method of claim 68, further comprisingadministering a termination regimen following the at least fourconsecutive treatment periods, wherein the termination regimen comprisesadministering to the subject, for a first period of 1-3 weeks, about 4mg of estriol; administering to the subject, for a second period of 1-3weeks following the first period, about 2 mg of estriol; and, after thesecond period, terminating administration of estriol to the subject. 70.The method of claim 69, wherein the method further comprises terminatingadministration of the norethindrone to the subject during the firstperiod of the termination regimen.
 71. The method of claim 68, whereinthe method comprises five consecutive treatment periods, six consecutivetreatment periods, seven consecutive treatment periods, or eightconsecutive treatment periods.
 72. The method of claim 68, wherein theestrogen and the progestrogen are formulated together.
 73. The method ofclaim 68, wherein the subject has a uterus.
 74. The method of claim 68,wherein the subject is a premenopausal female.
 75. The method of claim68, wherein the subject is a perimenopausal female.
 76. The method ofclaim 68, wherein the subject is experiencing progression of themultiple sclerosis.
 77. The method of claim 68, wherein the subject hasprogressive walking disability.
 78. The method of claim 68, wherein thesubject is a postmenopausal female.
 79. The method of claim 68, whereinthe method further comprises administering subcutaneously to thesubject, on a continuous basis for the treatment period, 20 mg ofglatiramer acetate daily,